Development of a Point-of-Care Immunochromatographic Test Based on Rhoptry Protein 14 for Serological Detection of Toxoplasma Gondii Infection in Swine

Background: Toxoplasma gondii, a worldwide distributed apicomplexan protozoan, can infect almost all warm-blooded animals and may cause toxoplasmosis. Globally, the prevalence of T. gondii in pigs varies between 10% and 60%. Therefore, the detection and prevention of swine toxoplasmosis is essential for agriculture and public health. In order to provide a point-of-care detection method for T. gondii infection in swine, an immunochromatographic test (ICT) was established. Methods: A sequence corresponding to amino acids 619-1061 of T. gondii rhoptry protein 14 (TgROP14) was cloned from T. gondii cDNA and expressed in Escherichia coli BL21 (DE3) strain. Monoclonal antibodies (mAb) against TgROP14 were prepared by immunizing BalB/c mice with the recombinant TgROP14 (rTgROP14), and the specicity, subtype, titer and concentration of the mAb were analysed. An immunochromatographic strip (ICT) based on puried rTgROP14, recombinant protein A and mAb against TgROP14 was developed. The specicity, sensitivity, and stability of this new ICT were evaluated. Finally, 436 porcine sera sampled from Zhejiang province in China were tested using the new ICT and a commercial IHA kit to assess the relative sensitivity and specicity. Results: rTgROP14 could be specically recognized by positive serum of T. gondii but not negative serum. The mAb TgROP14-5D5 shows higher specic recognition of T. gondii antigens and was therefore selected for subsequent colloidal gold strip construction. The new ICT based on TgROP14 exhibited good diagnostic performance with high specicity (89.6%) and sensitivity (100%). Among 436 eld porcine sera, ICT and IHA each detected 134 (30.7%) and 99 (87.5%) positive samples, respectively. The relative agreement was 87.5%. Conclusions: This new ICT based on TgROP14 is a candidate for routine testing of T. gondii


Background
Toxoplasma gondii is a worldwide distributed apicomplexan, which can parasitize nearly all types of warm-blooded animals including humans [1]. Approximately one third of human population globally has been infected by T. gondii [2]. T. gondii infection in healthy adults is usually asymptomatic; however, devastating consequences often occur in the congenital infections and the people of compromised immunity [3]. The majority of human T. gondii infections are caused by oral ingestion of raw or inadequately cooked meat containing viable tissue cysts, or by consuming food or water contaminated with cat-shed oocysts [4,5]. Pigs (Sus scrofa), as an intermediate host of T. gondii, have important economic and public health signi cance [6], and tissue cysts can survive for more than two years when most pigs are sacri ced as pork within a year [5]. Globally, the infection rate of T. gondii in pigs varies widely, typically between 10% and 60% [7]. China has one of the largest pork industries worldwide with the swine prevalence of T. gondii infection ranging from 58.1% in southern [8] to 24.5% in central [9]. Therefore, detection and prevention of swine toxoplasmosis is of great signi cance for agriculture and public health in China as well as the rest of the world.
Clinical manifestations of toxoplasmosis are not always obvious and speci c, diagnosis of T. gondii infection generally relies on laboratory testing of parasites, antibodies, and/or DNA [10] using pathogenic, immunological, imaging techniques and molecular detection [11]. Immunological diagnostics have been the preferred and the most commonly used [12]. They include indirect hemagglutination test (IHA), latex agglutination test (LAT) and enzyme-linked immunosorbent assays (ELISA) [13]. These methods are commonly used even though having certain limitations such as skillful operator, expensive equipment and time consuming. The colloidal gold immunochromatographic test (ICT) has been considered suitable for rapid detection outdoors, as it is simple to operate and no need for equipment plus small in size and convenient to store [14]. Several ICTs have been developed for detection of T. gondii infections, including based on surface antigen 2 (SAG2) [15], surface antigen 3 (SAG3) [16] or dense granule antigen protein 7 (GRA7) [17,18].
Rhoptry proteins of T. gondii (TgROPs) are potential diagnostic antigens [19,20,21,22]. Among them, TgROP14 locates to the parasitophorous vacuole membrane (PVM), possibly serving as a membrane transporter in participating in the exchange of substances between the parasitophorous vacuole (PV) and the host cell cytoplasm [23]. We aimed to use TgROP14 as a key molecule in a reliable, yet quick and user-friendly diagnostic method for detection of T. gondii infection that can be used at point-of-care.

Soluble protein preparations and western blot
Freshly harvested T. gondii tachyzoites, were washed thrice in phosphate buffered saline (PBS), sonicated 5 × 20 s at 5 kHz, and centrifuged at 12,000 g for 10 min to collect the supernatant containing T. gondii soluble antigens. About 10 µg soluble protein was loaded into each of lanes and transferred to 0.22 µm PVDF membranes (Millipore, Germany). Membranes were blocked with 5% (w/v) skim milk for 1 hour at room temperature and probed with appropriate primary antibodies, followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Fudebio, China) for 1 hour. Blots were exposed using ECL substrates (Fudebio, China) and visualized using the ChemiDoc™ chemiluminescence system (Bio-Rad, USA).
Preparation of the recombinant protein TgROP14 (rTgROP14-His) Following the manufacturer's instructions (Invitrogen, USA), total RNA of T. gondii RHΔku80Δhxgprt tachyzoites was extracted with TRIzol and was reverse-transcribed into cDNA using the reverse transcription kit ReverTra Ace-α-® (Toyobo, Japan). An insert corresponding to amino acids 619-1061 of TgROP14 (GenBank accession number DQ096565.1) was ampli ed by PCR using following primers: 5′-CCGGAATTCATGCCAGACCAGGTTATGGATTCAG-3′ and 5′-CCCAAGCTTCAGCGCTTGCTTCTTCCTAGTC-3′, including an EcoR I and a Hind III restriction enzyme site underlined. EcoR I-and Hind III-digested PCR products were cloned into the Escherichia coli expression vector pET-30a (Novagen, China). The right construct con rmed by sequencing was transformed into E. coli BL21 (DE3) (TaKaRa, Japan) to express rTgROP14-His by 1 mM IPTG induction at 37°C. After bacterial sonication in PBS (0.1 M, pH 7.4), rTgROP14-His was puri ed by Ni-NTA agarose (GE, USA). The quality of rTgROP14-His was veri ed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB).

Production of mouse monoclonal antibodies against TgROP14
For mAb preparation, six-week-old female BalB/c mice (Hangzhou, Zhejiang, China) were immunized with puri ed rTgROP14-His and then the immunized spleen cells of mice were isolated and fused with SP2/0 myeloma cells [24]. Brie y, for the rst immunization, each mouse received 100 µg of puri ed rTgROP14-His mix with an equal volume of the complete Freund's adjuvant. For the second and third immunization, the mice were immunized with the same dosage of puri ed rTgROP14-His mixed in a 1:1 ratio with the incomplete Freund's adjuvant, both at 2 weeks interval. One week after the third immunization, a booster intraperitoneal injection was given with only 50 µg rTgROP14-His. Three days afterwards, harvested spleen cells were fused with SP2/0 myeloma cells by polyethylene glycol.
Hybridomas were screened by ELISA for producing monoclonal antibodies (mAbs) against TgROP14 using rTgROP14-His to coat wells. ELISA-positive hybridomas were further screened by IFA. Brie y, T. gondii tachyzoites-infected Veros were xed in 4% paraformaldehyde (PFA) for 15 min at 4°C, permeabilized with PBS containing 0.25% Triton X-100 for 15 min at 37°C, and then blocked in 1% bovine serum albumin (BSA) for 1 h at 37°C. Subsequently, they were incubated both for 1 h in mAbs anti-TgROP14 (1:1000) and Alexa Fluor 488-conjugated secondary antibodies (1:1000) and stained with DAPI (4',6-diamidino-2-phenylindole). Finally, the uorescence images were obtained by a laser scanning confocal microscope (Zeiss LSM 780, Jena, Germany). Positive hybridomas identi ed were subcloned three times by limiting dilution method. Ascites was generated in the para n-primed BalB/C mice and puri ed using saturated ammonium sulfate [(NH 4 ) 2 SO 4 ]. The quality and speci city of the mAbs were tested by SDS-PAGE and western blot.
Identi cation of types and epitopes' speci cities of the mAbs A mouse monoclonal antibody isotyping kit (Biodragon, China) was selected to determine the isotype of the mAbs. The epitopes' speci cities of the two mAbs prepared were tested by an ELISA overlap experiment, and additivity indexes (A.I) of different mAbs combined were calculated using methods as described before [25].

Preparation of immunoassay materials
First, colloidal gold solution was prepared. Brie y, 2.5 mL of 1% trisodium citrate solution was supplemented into boiled 100 mL of 0.01% HAuCl 4 solution by stirring thoroughly and continuously. Kept the mixture boiling for an additional 10 min after its color turned from blue to dark red. Afterwards, constantly stirred the mixture for another 5 min before the preparation completed. The prepared gold colloids were stored in dark at 4°C with 0.01% (m/v) sodium azide (NaN 3 ). Transmission electron microscopy was used to identify the gold colloids.
The desalted recombinant protein rTgROP14-His was used as an antibody detector after being conjugated with the colloidal gold, while staphylococcal protein A (Sangon Biotech, Shanghai) was used as the capture protein. The optimum conditions were determined as follows: 0.2 mL of puri ed and desalted rTgROP14-His (1.5 mg/mL) was supplemented into 20 mL of colloidal gold solution (pH 8.2). The mixture was stirred carefully for 15 min, and blocked by 10% BSA (m/v) for 1 hour. After centrifugation (12,000g, 30min), the colloidal gold-antigen conjugate from the sediment was resuspended in 2 mL dilution buffer [0.2 M tris solution (pH 8.0) with 10% BSA, 20% Sucrose, 5% trehalose and 0.2% NaN 3 ] and stored at 4°C. The recombinant protein A (4 mg/mL) and TgROP14-5D5 mAb (2 mg/mL) were transferred to the nitrocellulose (NC) membrane (Millipore, China) with a rate of 1 µL/cm, respectively forming the test and control lines. The strips were incubated and dried at 37°C for 1 h.

Preparation of the immunochromatographic strip
The ICT strip was assembled as showed in Fig. 1. It consisted of a sample pad, which saturated with 0.01 M PBS (pH 8.2) containing 0.1% Tween-20, a conjugate pad which was added with colloidal gold probe, an immobilized NC membrane, and an absorbent pad. Both sample pad and the conjugate pad along with the NC membrane prepared above were dried at 37°C. Pure cellulose ber was served as the absorbent pad and the PVC plate was set as the assay membrane at the bottom of the test strip. These strips were sequentially overlapped with the sample pad, conjugate pad, xed NC membrane and absorbent pad, cut into 4 mm width and stored with desiccators at 4°C until use. Sensitivity, speci city, and stability of the immunochromatographic test To identify the detection limit of ICT, the standard T. gondii-positive porcine serum were diluted in a ratio of 1:2, 1:4, 1:8 with 0.9% NaCl (pH 7.2), and the standard negative porcine serum as the negative control. The speci city of ICT was evaluated using positive porcine sera for the porcine reproductive and respiratory syndrome (American type), the swine type O foot-and-mouth disease, the swine type A footand-mouth disease and the swine fever conserved in our laboratory. The strips stored at 4°C for 12 weeks were used to examine the stability of this ICT.
Detection of T. gondii infection in eld samples 436 porcine sera sampled from Zhejiang province in China were tested using the new ICT. They were also detected by the IHA kit (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, China) serving as a "reference standard" to assess the relative sensitivity and speci city of the newly developed ICT.

rTgROP14-His proteins are recognized by antibodies to T. gondii
The recombinant protein rTgROP14-His was generated by transfecting E. coli BL21 (DE3) with the plasmid pET30a-TgROP14-His. It was approximately 70 kDa as con rmed by SDS-PAGE (Fig. 2a) and Western blot with anti-His antibody (Fig. 2b). Further, rTgROP14-His was recognized by mouse polyclonal antibodies to T. gondii (Fig. 2c), which indicated that rTgROP14-His has good immunogenicity.
TgROP14-5D5 is the candidate for colloidal gold strip Two mAbs 1E9 and 5D5 against rTgROP14-His were characterized using the natural antigens of T. gondii tachyzoites by Western blot. Both detected two proteins at approximately 140 and 70 kDa (Fig. 3a), and it is possible that the upper band is the dimer of the lower band, alternatively, they were the same proteins with different post-translational modi cations. They were further determined to be IgG3 and IgG2a, respectively ( Table 1). The two mAbs were successfully puri ed by saturated ammonium sulfate as shown by the heavy chain (∼55 kDa) and the light chain (∼25 kDa) in SDS-PAGE (Fig. 3b). Furthermore, the value of A.I was 22.8%, which demonstrated that these two mAbs targeted the same epitope (Table  1). Speci cities of these two mAbs were further analysed by IFA, and TgROP14-5D5 shows higher speci c recognition of T. gondii antigens than TgROP14-1E9 (Fig. 3c). TgROP14-5D5 had a titer of about 1:3.3×10 7 , and was selected for subsequent colloidal gold strip construction because of its higher speci city. Immunochromatographic test using TgROP14-5D5 is sensitive and speci c The sensitivity of the ICT was gauged by a series of 1:2 diluted T. gondii positive porcine serum samples up to 1:16. The red color at the testing line can be clearly observed when the sera were undiluted to 1:4 ( Fig. 4a), indicating that the detection limit of the ICT was 1:4. The speci city of the ICT was veri ed with porcine serum samples positive for 4 different pathogens which were often clinically observed. They included porcine reproductive and respiratory syndrome virus, swine type O and A foot-and-mouth disease virus and swine fever virus. Two red lines were only observed with the T. gondii positive porcine serum at the test and control zones. In contrast only the control line appeared when each of other sera was tested (Fig. 4b). Furthermore, the red line could be clearly seen in the test band exposed to positive porcine serum with the same batch of ICT strips after stored at 4°C for 12 weeks (data not shown), indicating that the test strips stored at 4°C were stable for at least 12 weeks.

Newly developed immunochromatographic test is suitable for eld samples
We next compared the newly developed ICT test with a commercially available IHA as a "gold standard".
We used 436 porcine sera collected from farm pigs in Zhejiang, China. The new ICT found 134 positive sera, which was 30.7%. In contrast, IHA detected 99 positive sera, representing 22.7% of all samples ( Table 2). The relative sensitivity and speci city of this newly prepared ICT were 100% and 89.6%, respectively, and the relative agreement between ICT and IHA was 87.5%.

Discussion
Toxoplasma lysate antigens (TLAs), prepared from mice and/or tissue culture derived tachyzoites, were used to develop early commercial serological detection kits for diagnosis of T. gondii [26]. However, the methods used to produce these antigens vary signi cantly between laboratories or even from batch to batch in the same lab. It is important to point out that TLAs prepared from tachyzoites also contain a variety of nonparasitic substances derived from host cells. Furthermore, low productivity and potential health biohazard of native antigens cannot be ignored. Therefore, the native antigens have been gradually replaced by recombinant antigens, which eliminate the risk of laboratory infection caused by working with live tachyzoites. Advantages of using recombinant proteins include easier to be standardized, ner repeatability, and consistency of protein preparation. Over the past 30 years, many recombinant antigens of T. gondii as diagnostic targets have been assessed to detect speci c antibodies in human serum [10,27]. Some researchers also evaluated the feasibilities of utilizing recombinant T. gondii antigens for the detection of speci c antibodies in animal sera [28,29]. To date, all recombinant antigens tested in animals were obtained by the E. coli prokaryotic expression of T. gondii proteins such as SAGs, microneme proteins (MICs), rhoptry neck proteins (RONs) and ROPs, and GRAs [30].
ROPs of T. gondii such as ROP1 and ROP2 have been used as detection antigens for diagnosis [19,20,21,22]. The recently discovered ROP14 may be a membrane transporter and likely to be located to the PVM, and participate in substance exchange between T. gondii and host cell [23]. In this study, the DNA sequence encoding TgROP14 minus transmembrane domain was PCR ampli ed and cloned, the resultant recombinant protein rTgROP14-His was obtained by the E. coli prokaryotic expression system. Further analyses rmly established that the rTgROP14-His was a potential candidate to be a diagnostic antigen.
Several serologic kits are commercially available for the diagnosis of T. gondii infection, including LAT, modi ed agglutination test (MAT), Western blotting test, and immuno uorescence antibody test (IFAT). All use native antigens. At present, most serologic methods require technical training and are laborious, and are hard to be used at the point-of care. ICT is not only highly sensitive and speci c, but also rapid, cost-e cient and of very minimum training requirement of operators, making it applicable and attractive for eld application [31]. Here, we developed an ICT using puri ed rTgROP14, recombinant protein A and mAb TgROP14-5D5 for serological detection of T. gondii in swine populations.
Several ICTs have been developed for detecting T. gondii infections so far. An ICT based on recombinant TgSAG2 was established for detection of anti-T. gondii antibodies in cat with relative sensitivity and speci city of TgICT of 100 and 94.5% compared with LAT and 97.2 and 95.8% compared with ELISA [15]. An additional ICT based on GRA7 was developed for detecting T. gondii infection in swine populations. The relative sensitivity and speci city were 80% and 100% when iELISA was regarded as a reference [17].
Previously, the sensitivity and speci city of a few other approaches were computed respectively: 45.9 and 96.9% for LAT, 82.9 and 90.29% for MAT, 29.4 and 98.3% for IHAT, and 72.9 and 85.9% for ELISA [32]. Compare with the above methods, the ICT developed in this study exhibited higher sensitivity but lower speci city. Current rule is that the authenticity of positiveness for T. gondii requires to be determined by at least two different commercial tests (e.g., MAT and ELISA), and the results acquired from a new diagnostic method such as using a recombinant antigen are compared with those of commercial tests. By this rule, the novel ICT based on rTgROP14 and TgROP14-5D5 mAb is promising to become a practical serological diagnostic test for the clinical investigation of T. gondii infection at the point-of-care.

Conclusions
In this study, we showed that the rhoptry protein TgROP14 can recognize positive serum of T. gondii but not negative serum. The mAb TgROP14-5D5 can speci cally recognize T. gondii antigens. The ICT using puri ed rTgROP14, recombinant protein A and mAb TgROP14-5D5 had good speci city and sensitivity.
The novel ICT has potential for serological detection of T. gondii at the point-of-care in swine populations.  Figure 1 Page 16/16

Figures
The component and illustration of the ICT strip.

Supplementary Files
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